Abstract
Lipases are very important biocatalysts in lipid modification and a broad number of processes have already been established on large scale in industry. A current trend is to tailor-design enzymes for a given application and protein engineering methods are commonly used for this. In this dossier, basic principles of rational protein design and directed evolution are described together with recent examples for the successful application of these tools for the alteration of the substrate specificity, stereoselectivity, and stability of lipases.
Highlights
Lipases (EC 3.1.1.3, triacylglycerol hydrolases) are the most widely used biocatalysts for the modification of fats and oils [1,2,3], but they are employed in organic synthesis [4, 5]
The majority of examples from academic research and in industrial applications rely on commercially available lipases and use just a few types, which all originate from microorganisms (i.e. lipase from Rhizomucor miehei (Lipozyme RM IM), Thermomyces lanuginosa (Lipozyme TL IM), Candida antarctica type B (Novozyme 435, CAL-B) or Burkholderia cepacia (Amano PS))
A two-step lipasecatalyzed process was developed, in which tripalmitin is first subjected to alcoholysis with ethanol using a lipase from Rhizopus delemar immobilized on a polypropylene carrier (EP-100) yielding 95% monopalmitin with a purity >90% after crystallization
Summary
Lipases (EC 3.1.1.3, triacylglycerol hydrolases) are the most widely used biocatalysts for the modification of fats and oils [1,2,3], but they are employed in organic synthesis [4, 5]. STAG containing medium chain fatty acids at the sn1- and sn3-position and a long (preferentially polyunsaturated) fatty acid at the sn2-position are used to treat patient with pancreatic insufficiency and for rapid energy supply (i.e. for sports) Another important example is BetapolTM used in infant nutrition, which contains oleic acid at the sn1and sn3- and palmitic acid at the sn2-position. It often occurs that an enzyme does not meet the requirements for a certain application and its properties have to be optimized This usually includes the chemo-, regioand stereoselectivity of the biocatalyst, and process-related aspects such as long-term stability at high temperatures or pH-values and activity in the presence of large substrate concentrations need to be improved. Molecular modeling site-directed mutagenesis A cloning and expression non-recombining epPCR saturated mutagenesis mutator strain SeSaM recombining DNA-(family)-shuffling StEP random-priming ITCHY/SCRATCHY
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